Mullerian Inhibiting Substance (MIS) is expressed in the developing male gonad and causes the regression of Mullerian ducts, the progenitor tissue for the uterus, fallopian tubes and upper vagina in females. Since SRY, a gene on the Y chromosome, has been implicated as the testis determining factor in mammals, it seemed likely that SRY plays a fundamental role in the expression of MIS. Accordingly, it has been shown that a luciferase reporter construct driven by the human MIS promoter (-114/+10) can be activated by expressed human SRY in tissue culture. Transfection studies with deletion constructs of the MIS promoter showed that activation by expressed SRY was observed with all constructs including one which contained only the initiator region of the MIS gene (-22/+10). To determine the nature of the activation of the MIS initiator by SRY we will address the following: Specific Aim 1. Determine whether activation of MIS by SRY involves direct interaction with the MIS sequence. This will be done by gel-mobility retardation assays with overexpressed SRY and assaying for the presence of SRY in the protein DNA complex. This will be accomplished by producing a fusion protein which has an immunodetectable tag. Specific Aim 2. Identify proteins which are interacting with SRY by using the yeast interaction trap assay. Since it is possible that SRY relies on protein-protein interactions for its ability to activate transcription, we will investigate whether other proteins implicated in human sex reversal interact directly with SRY. We will also screen for novel interactors from a developmentally staged library.